The proteasome plays a central role in maintaining cell viability in response to stressors, such as protein overload or endoplasmic reticulum (ER) stress, by regulating signal transduction that promotes cell survival (i.e., NF-?B) and degrading potentially cytotoxic proteins (i.e., defective ribosomal products or DRiPs). The proposed work investigates the interplay between specific stressors (Protein Overload and ER Stress) and the immunoproteasome by defining conditions that induce immunoproteasome expression and testing specific hypotheses regarding immunoproteasomes role in responding to cellular stress. While its role in generating immunogenic peptides for antigen presentation has been clearly established, the expression of immunoproteasome subunits in non-inflammed immune-privileged tissue such as retina and brain implies other non-immune functions are possible. Notably, the upregulation of immunoproteasome in diseased retina and brain suggests it responds to challenges that induce stress and injury. The central hypothesis is that the immunoproteasome is a key component of the cellular response to stress. We have made several predictions that can be experimentally tested using techniques and strategies already familiar to our lab and the labs of our collaborators using knock-out mice lacking two (lmp7-/-/mecl-1-/-, L7M1) catalytic subunits of the immunoproteasome and conditional SCA1 transgenic mice expressing either the WT [30Q] or the mutant [82Q] ataxin-1 protein. Cell lines generated from WT and L7M1 mice and transfected with either the WT [30Q] or mutant [82Q] ataxin protein provide excellent in vitro model systems for testing our hypotheses. (Aim 1) Hypothesis: Immunoproteasome is required for effective muscle remodeling. Muscle remodeling will be induced by hindlimb unweighting WT and L7M1 mice to test if immunoproteasome (a) helps maintain muscle function during muscle remodeling, (b) provides protection from cellular damage, and (c) is directly involved in muscle remodeling. (Aim 2) Hypothesis: Immunoproteasome helps protect cells from the toxic effects of misfolded proteins. Using the conditional SCA1 transgenic mouse and cultured cells (WT and immunoproteasome-deficient) transfected with the WT [30Q] or mutant [82Q] ataxin protein, we will test if (a) the presence of a mutant, misfolded protein induces expression of the immunoproteasome, and (b) immunoproteasome provides protection from misfolded proteins. (Aim 3) Hypothesis: Reduced immunoproteasome expression increases the susceptibility to ER stress-induced damage. Using WT and immunoproteasome-deficient cell lines, we will test if immunoproteasome (a) is upregulated under conditions of ER stress, (b) protects from ER stress, and (c) immunoproteasome is involved in activation of the stress- sensitive transcription factor NF-?B. The proposed research brings together a powerful team with expertise in proteasome biology (Ferrington), muscle physiology (Thompson), and age-onset polyglutamine diseases (Orr). This collaboration incorporates techniques in molecular genetics, cell biology, biochemistry, and muscle physiology in pursuing evidence consistent with novel roles for the immunoproteasome in protecting the cell from a variety of stressors. PROJECT NARRATIVE: The proposed work investigates the interplay between specific stressors (Protein Overload and ER Stress) and the immunoproteasome, a specialized proteasome subtype, by defining conditions that induce immunoproteasome expression and testing specific hypotheses regarding immunoproteasomes role in responding to cellular stress. The approach is to use knock-out mice lacking two (lmp7-/-/mecl-1-/-, L7M1) catalytic subunits of the immunoproteasome and conditional SCA1 transgenic mice expressing either the wildtype [30Q] or the mutant [82Q] ataxin-1 protein. Cell lines generated from WT and L7M1 mice and transfected with either the WT [30Q] or mutant [82Q] ataxin protein provide excellent in vitro model systems for testing our hypotheses.